Phagocytosis or engulfment, is a specialized form of endorytozis through which eukaryotes take up very lag particles, or even whole Cells. It is a fundamental biological process conserved from single-cell organisms, such as amoebae to mammals (Metchnikoff, E. 1891), Lectures on the comparative pathology of inflammation; delivered at the Pasteur Institute, 1891, 1968 Edition (New York: Dover Publication)), Initially used for the dual purpose of feeding and defense, phagocytosis evolved, following the emergence of mesoderm, into a mechanism used to protect the host against invading organisms and to clear up foreign particles and cell debris (Metchnikoff, 1891). Recently, the significance of phagocytosis has been extended due to its role in eliminating cells undergoing programmed cell death (apoptosis), Since apoptosis has been implicated in a number of human diseases elucidation of the regulation of this phagocytosis is highly desirable since it may lead to a new route of therapeutic intervention in these diseases.
Genetic studies in C. elegans have identified over a dozen genes that function in programmed cell death. The present inventors have used the positional method to clone and have functionally characterized the C. elegans gene ced-6. It is shown that the CED-6 protein contains a phosphotyrosine binding domain and several potential SH3 binding sites. It is further demonstrated that CED-6 acts within engulfing cells, and functions to promote the removal of both early and persistent cell corpses. Overexpression of ced-6 can partially suppress the engulfment defect of both ced-1 and ced-7, suggesting that ced-6 might function downstream of these two genes. It is concluded that CED-6 acts as an adaptor molecule in a signal transduction pathway that mediates the engulfment of apoptotic cells in C. elegans. The present inventors have also identified isolated and characterized human ced-6 homologue including a splice variant thereof, which it is shown is involved in a similar process in mammalian cells.
The invention provides, in isolated form, a protein which is the CED-6 protein of C. elegans or a protein which has equivalent function thereto and two human homologues of said protein, hereinafter referred to as h1 CED-6 and h2CED-6.
The invention further provides a functional fragment of CED-6, h1CED-6 and h2CED-6 for example, a fragment corresponding to the phosphotyrosine binding domain and/or the proline/serine rich region.
The invention further provides an isolated nucleic acid encoding CED-6 and two human homologues of CED-6, as well as nucleic acid encoding functional fragments of CED-6, h1CED-6 and h2-CED-6 as described above.
The invention further provides nucleic acid which is antisense to any of the nucleic acids described above or which is capable of hybridizing to any of the nucleic acids described above under conditions of low stringency or portions or fragments thereof.
The invention further provides expression vectors comprising nucleic acid encoding CED-6, h1CED-6, h2CED-6 or encoding functional fragments of said proteins as above.
The invention further provides mammalian cell-lines transfected with one or more nucleic acids encoding CED-6, h1CED-6 and h2CED-6.
The invention further provides assay methods using the proteins, nucleic acids and transfected cells described above to identify compounds which enhance or inhibit the signal transduction pathway in which CED-6, h1CED-6 and/or h2CED-6 participate.
The invention further provides assay methods using the transfected cells described above to identify compounds which enhance or inhibit the expression of the ced-6, h1ced-6 or h2ced-6 genes.
The invention further provides antibodies which react with an epitope of CED-6, h1CED-6 and/or h2CED-6.
The invention further provides a method of treating diseases the etiology of which may be attributed to failure of engulfment of apoptotic or other diseased cells such as inflammation autoimmune disease or cancer by administering to a patient one or more of the aforesaid proteins or nucleic acids or compounds which are enhancers of CED-6, h1CED-6 or h2CED-6.
The invention further provides a method of treating diseases which would benefit from a reduction in the engulfment of apoptotic cells, such as, neurodegenerative diseases stroke, or sickle-cell anaemia, by administering one or more of the aforesaid proteins, nucleic acids or compounds which are inhibitors of CED-6, h1CED-6 or h2CED-6.
The invention further provides a method of diagnosis of a human or animal disease using a nucleic acid encoding CED-6, h1 CED-6 or h2CED-6 or the complement thereof or an antibody to CED-6, h1CED-6 or h2CED-6 to detect a genetic defect.
The invention further provides a method of identifying proteins which interact with CED-6, h1CED-6 or h2CED-6 in the signal transduction pathway in which those proteins participate.
The invention further provides a fusion protein in which CED-6, h1CED-6 or h2CED-6 or a functional fragment thereof such as the phosphotyrosine binding domain or serine proline rich region, is fused to another protein such as an epitope tag or product of a reporter gene.
The invention further provides a method of determining whether a compound is an enhancer or Inhibitor of the signal transduction pathway in which CED-6 participates by observing the effect of the compound on C. elegans worms having altered CED-6 expression.